TWI875469B - Magnetic bead based nucleic acid extraction system - Google Patents

Abstract

A magnetic bead based nucleic acid extraction system includes a plurality of magnetic beads and an alcohol-free buffer system. The alcohol-free buffer system includes a lysis binding buffer, a first wash buffer, a second wash buffer, and an elution buffer that are stable in storage at room temperature. The buffers include a special selection of chaotropic salt, detergent, precipitant and other necessary components for ideal extraction of nucleic acids. The magnetic bead based nucleic acid extraction system is stable, user-friendly and environment-friendly, efficient and efficacious nucleic acid extraction system optimal for pathogens from swab samples, and can achieve efficient both DNA and RNA extractions, be it separately or simultaneously, from both virus and bacteria in swab samples.

Description

Magnetic Bead Nucleic Acid Extraction System

This case relates to a nucleic acid extraction system, in particular to a magnetic bead-based nucleic acid extraction system.

Nucleic acid detection has become the most important molecular diagnostic technology in recent years due to its excellent sensitivity and specificity, and is widely used in various fields such as genetic disease research, oncology, and infectious disease epidemiology. The prerequisite for sensitive detection of the target lies in nucleic acid extraction. The main function of nucleic acid extraction is to obtain high-quality nucleic acids and remove inhibitory factors present in the sample matrix. Many extraction methods have been developed based on different extraction mechanisms, and existing methods can be roughly divided into two categories: chemically driven methods and solid phase based methods.

Chemically driven methods are early developments that utilize phase separation of phenol-chloroform in solution and precipitation of nucleic acids with alcohols such as isopropanol and ethanol. Cell lysis occurs in the presence of strong protein denaturing agents such as sodium dodecyl sulfate (SDS) and proteinase K, followed by the addition of a mixture of phenol:chloroform:isoamyl alcohol (25:24:1) to promote phase separation of DNA, lipids, and other cellular debris, with DNA in the aqueous phase and the rest in the organic phase. Under centrifugal force, the aqueous phase containing the DNA is located above the organic phase and can be transferred to a clean tube for analysis or further recovered and concentrated by ethanol precipitation. However, this method has several disadvantages. First, it is very time-consuming and labor-intensive. Furthermore, these procedures carry a high risk of causing harm to operators due to the use of carcinogenic and toxic chemicals such as chloroform or phenol. In addition, there is a risk of phenol contamination of the extracted nucleic acid, and the probability of nicking at many sites of the nucleic acid is increased, which seriously affects the stability of the extracted nucleic acid. In addition, ethanol causes inconvenience and high cost in reagent transportation and storage.

The salting-out method is an alternative to the phenol-chloroform extraction procedure. It is based on the principle that proteins and other cellular contaminants will precipitate in saturated salt solutions due to their relative hydrophobicity, while DNA will not. After cell lysis, a saturated salt solution, such as 6 M sodium chloride (NaCl), is added. The well-mixed cell lysate-salt solution is then centrifuged to precipitate the protein components. The supernatant contains the DNA, which can be transferred to a new tube and mixed with 2 volumes of pure ethanol to further precipitate and purify the DNA. This method avoids the use of toxic organic chemicals. However, it is not uncommon that the salt used for protein precipitation may also contaminate the extracted nucleic acids, which in turn inhibits nucleic acid amplification by enzymes, such as PCR or other isothermal amplification methods.

The selective binding of nucleic acids using solid phases of various materials is a later development that allows single-vessel nucleic acid extraction compared to traditional extraction methods based on phase separation and salt separation mechanisms using multiple vessels. Since the solid phase function is affected by the contact solution, whether it is lysis buffer, binding buffer, washing buffer or rinse buffer, the buffer formulation is also an important part of an efficient nucleic acid extraction system. The solid phase used to bind nucleic acids can be carrier materials, spin filters, silica particles, cuvettes, microtiter plates, filter membranes, polystyrene beads and nitrocellulose paper, and many methods have been developed accordingly.

For the above methods, whether based on liquid phase separation or solid phase separation, a centrifuge is a necessary device, which hinders the high automation of sample preparation. The latest development of nucleic acid extraction is that magnetic beads can be functionalized with different functional groups to selectively bind nucleic acids, achieving rapid and highly automated nucleic acid extraction.

Convenient and effective nucleic acid extraction has made technical progress in biological and molecular sciences through kits consisting of special devices and related buffers, but there are still some challenges to be solved. First, most commercial nucleic acid extraction kits require the end user to provide a list of consumables and/or equipment, which can only be met in well-equipped laboratories. Secondly, in the above-mentioned newly developed technology of using functionalized magnetic beads as carriers to specifically capture nucleic acids, alcohol or isopropanol is an important component of the buffer. When placed on a cartridge for point of care (POC) applications, alcohol reacts with the plastic, thereby destroying its mechanical/physicochemical properties, and also causes problems due to its volatility, flammability and potential for leakage. These properties make alcohol a highly regulated substance by the International Air Transport Association (IATA). Third, the turn-around time of most commercial kits exceeds half an hour. Fourth, most extraction buffers can only be used to extract DNA or RNA from viruses or bacteria alone, and there is a lack of a universal buffer system that can effectively extract and purify all nucleic acids from pathogens in human swab samples. Finally, some special components in the extraction buffer require freezing/refrigeration at -20°C or 4°C to obtain the required performance, especially those kits containing proteases for cell lysis and nuclease denaturation and vector nucleic acids for low template input, thus causing inconvenience in transportation and storage.

Therefore, it is necessary to provide a nucleic acid extraction system to solve the challenges encountered in the prior art.

One of the purposes of the present invention is to provide a stable, user-friendly, environmentally friendly, effective and efficient magnetic bead-based nucleic acid extraction system suitable for pathogens in swab samples.

Another object of the present invention is to provide an alcohol-free magnetic bead-based nucleic acid extraction system to avoid the problems caused by alcohol.

Another object of the present invention is to provide a magnetic bead-based nucleic acid extraction system that can effectively extract DNA and RNA from viruses and bacteria in swab samples individually or simultaneously.

To achieve the above object, the present invention provides a magnetic bead-based nucleic acid extraction system, comprising a plurality of magnetic beads and an alcohol-free buffer system. The alcohol-free buffer system comprises a lysis-binding buffer, a first washing buffer, a second washing buffer, and an extraction buffer. The lysis-binding buffer comprises 0.5 to 4 M guanidine thiocyanate, 5 to 20% (v/v) PEG8000, 10 to 1000 mM sodium citrate, and a non-ionic detergent selected from the group consisting of 0.5 to 4% (v/v) NP-40 substitute, 0.5 to 4% (v/v) Tergitol 15-S-40, 0.5 to 4% (v/v) Ecosurf EH-9, and a mixture thereof. The first washing buffer includes 0.5 to 3 M guanidine thiocyanate, 5 to 20% (v/v) PEG8000, 0.5 to 2 M sodium chloride, 5 to 25 mM EDTA, and a non-ionic detergent selected from the group consisting of 0.25 to 4% (v/v) Ecosurf EH-9, 0.1 to 4% (v/v) Triton X-100, 0.25 to 4% (v/v) Tergitol 15-S-40, and mixtures thereof. The second washing buffer includes 1 to 25 mM hexaaminecobalt (III) chloride, 5 to 20% (v/v) PEG8000, 5 to 1000 mM sodium chloride, and a non-ionic detergent selected from the group consisting of 0.25 to 2% (v/v) Ecosurf EH-9, 0.1 to 2% (v/v) Triton X-100, 0.25 to 2% (v/v) Tergitol 15-S-40, and mixtures thereof.

In one embodiment, the pH of the lysis-binding buffer is between 3 and 5.

In one embodiment, the pH value of the first cleaning buffer is 3 to 5.

In one embodiment, the first cleaning buffer further comprises 0.01 to 1% (v/v) lactic acid.

In one embodiment, the pH value of the second cleaning buffer is 3 to 5.

In one embodiment, the second cleaning buffer further comprises 0.01 to 1% (v/v) lactic acid.

In one embodiment, the pH of the flushing buffer is 8.

In one embodiment, the wash buffer comprises 1 to 25 mM EDTA.

Some embodiments that embody the features and advantages of the present invention will be described in detail in the following description. It should be understood that the present invention can have various variations in different aspects without departing from the scope of the present invention, and the descriptions and drawings are essentially for illustrative purposes rather than for limiting the present invention.

The present invention provides a stable, user-friendly, environment-friendly, effective and efficient total nucleic acid extraction and purification system suitable for both viruses and bacteria in swab samples. The system includes a buffer, magnetic beads and a protocol for extracting nucleic acids from swab samples. The system is capable of lysing the starting material, binding the nucleic acids to the magnetic beads, washing impurities and extracting the nucleic acids from the magnetic beads. The relevant extraction protocol has also been optimized to achieve effective extraction of DNA and RNA from pathogens in swab samples either separately or simultaneously.

Accordingly, the present invention provides a nucleic acid extraction and purification system that uses a solid phase mechanism, particularly magnetic separation. Magnetic separation uses magnetic beads, preferably paramagnetic beads, to extract nucleic acids from cell lysates. The principle is that by adjusting the buffer conditions, the magnetic beads and nucleic acids can be reversibly bound. Magnetic separation does not require a centrifugation step, and does not require the removal of supernatant or replacement of containers, so a fully automated extraction process can be achieved.

In traditional magnetic separation techniques, alcohol is used in most cleaning buffers, and in some protocols is also used in lysis buffers or elution buffers. However, due to its volatility, flammability, possibility of leakage, and restrictions on the transportation of alcohol in many countries, the presence of alcohol creates problems. Therefore, the buffer system of the present case is designed to be alcohol-free, thereby avoiding the problems caused by alcohol. In short, the magnetic bead-based nucleic acid extraction system of the present case includes a plurality of magnetic beads and an alcohol-free buffer system. The buffer system includes a lysis binding buffer, a wash buffer, and an elution buffer, which will be described in detail as follows.

Lysis and binding buffer is used to lyse samples and bind nucleic acids to magnetic beads. This is achieved through the combination of detergents, chaotropic salts, precipitants, and salts with optimal pH.

Detergents can destroy the structure of lipid biolayers and some proteins in microbial membranes, thereby destroying the integrity of the membrane and promoting cell lysis. According to the different molecular structures, detergents can be divided into four types: anionic detergents, cationic detergents, non-ionic detergents and zwitter ionic detergents. In this case, the lysis and binding buffer used is a non-ionic detergent.

In one embodiment, the non-ionic cleaner is selected from the group consisting of NP-40 alternative (NP-40 alternative, CAS number 9016-45-9, and does not contain NP-40), Tergitol 15-S-40 (secondary alcohol ethoxylate, CAS number 84133-50-6), Ecosurf EH-9 (2-ethylhexan-1-ol; 2-methyloxirane; oxirane, CAS number 64366-70-7) and mixtures thereof, wherein the concentration of NP-40 alternative is 0.5 to 4% (v/v), preferably 0.8 to 1% (v/v); Tergitol The concentration of 15-S-40 is 0.5 to 4% (v/v), preferably 0.5 to 1.25% (v/v); the concentration of Ecosurf EH-9 is 0.5 to 4% (v/v), preferably 0.5 to 1.25% (v/v). This case provides three sets of lysis-binding buffers, which are prepared from NP-40 substitute, Tergitol 15-S-40, or Ecosurf EH-9.

Isolating salts can denature proteins because they have the ability to destroy hydrophobic interactions. Isolating salts help lyse cell membranes, inactivate nucleases, and promote the binding of nucleic acids to magnetic beads. In one embodiment, guanidine thiocyanate (GuSCN) is one of the most effective lysing agents and is used in the lysis and binding buffer of this case, and the concentration of GuSCN is 0.5 to 4M, preferably 1 to 3M.

DNA or RNA precipitation is an important step in separating nucleic acids from other components of a sample. It can be done by a variety of mechanisms, the two most prominent of which are reducing the repulsive forces between these macromolecules, as in the case of multivalent cations, and weakening the interaction between nucleic acids and water molecules. Alcohol is an effective precipitant to reduce the interaction between nucleic acids and water molecules, but it has the problems mentioned above.

In the present case, the lysis and binding buffer uses a non-alcoholic precipitant. In one embodiment, the precipitant includes a high molecular weight PEG8000 at a concentration of 5 to 20% (v/v), preferably 5 to 10% (v/v), and a sodium citrate at a concentration of 10 to 1000 mM, preferably 10 to 50 mM, to effectively concentrate the nucleic acids in the sample, and the latter also helps to chelate metal ions that can activate DNase and RNase. In some embodiments, PEG8000 can be replaced by other PEGs with different molecular weights.

In addition to the rational selection of chemicals and their optimal concentration, another important factor is the pH of the buffer. Specially manufactured magnetic beads have functional groups on their surface that can specifically attach and separate nucleic acids, and this function depends on the pH of the buffer. The pH value of the lysis-binding buffer is 3 to 5, and is provided by tris-acetate, with a concentration of 5 to 100 mM, preferably 5 to 20 mM, and the magnetic beads have the highest binding capacity at this concentration.

Therefore, as described above, the lysis and binding buffer used in the magnetic bead-based nucleic acid extraction system of the present invention includes 0.5 to 4 M GuSCN, 5 to 20% (v/v) PEG8000, 10 to 1000 mM sodium citrate, and a non-ionic detergent selected from the group consisting of 0.5 to 4% (v/v) NP-40 substitute, 0.5 to 4% (v/v) Tergitol 15-S-40, 0.5 to 4% (v/v) Ecosurf EH-9 and a mixture thereof. The pH value of the lysis and binding buffer is adjusted with concentrated acetic acid. The present invention provides three sets of lysis and binding buffers A, B, and C, and their formulas are shown in Tables 1 to 3 below.

Table 1: Lysis Binding Buffer A Element Concentration range Tris-acetate (pH 3 to 5) 5 to 100 mM G JZ 0.5 to 4 M PEG 8000 5 to 20% (v/v) Ecosurf EH-9 0.5 to 4% (v/v) Sodium citrate 10 to 1000 mM

Table 2: Lysis Binding Buffer B Element Concentration range Tris-Acetate (pH 3 to 5) 5 to 100 mM G JZ 0.5 to 4 M PEG 8000 5 to 20% (v/v) NP-40 alternatives 0.5 to 4% (v/v) Sodium citrate 10 to 1000 mM

Table 3: Lysis Binding Buffer C Element Concentration range Tris-Acetate (pH 3 to 5) 5 to 100 mM G JZ 0.5 to 4 M PEG 8000 5 to 20% (v/v) Tergitol 15-S-40 0.5 to 4% (v/v) Sodium citrate 10 to 1000 mM

The washing buffer is used to remove impurities present in the sample from the nucleic acid. Since the nucleic acid is bound to the magnetic beads, the washing buffer must destroy the nucleic acid bound to the magnetic beads to the minimum extent when removing the impurities. The magnetic bead-based nucleic acid extraction system of the present case uses at least two washing buffers, each of which includes a detergent, a separation salt, a precipitant, a chelating agent, and a salt with an optimal pH value.

In this case, the cleaning buffer uses a non-ionic cleaner to help remove impurities. In one embodiment, the non-ionic cleaner is selected from the group consisting of Ecosurf EH-9, Triton X-100, Tergitol 15-S-40 and mixtures thereof. In the first cleaning buffer of the present invention, the concentration of Ecosurf EH-9 is 0.25 to 4% (v/v), preferably 0.5 to 1.25% (v/v); the concentration of Triton X-100 is 0.1 to 4% (v/v), preferably 0.1 to 2% (v/v); and the concentration of Tergitol 15-S-40 is 0.25 to 4% (v/v), preferably 0.5 to 1% (v/v). In the second cleaning buffer of the present invention, the concentration of Ecosurf EH-9 is 0.25 to 2% (v/v), preferably 0.25 to 0.75% (v/v); the concentration of Triton X-100 is 0.1 to 2% (v/v), preferably 0.1 to 1% (v/v); the concentration of Tergitol 15-S-40 is 0.25 to 2% (v/v), preferably 0.25 to 0.75% (v/v). The present invention provides three sets of cleaning buffers, which are prepared from Triton X-100, Tergitol 15-S-40 or Ecosurf EH-9 respectively.

In one embodiment, the pH value of the washing buffer is 3 to 5, provided by Tris-HCl at a concentration of 10 to 100 mM, which is most suitable for maintaining the binding of nucleic acids to magnetic beads. Optionally, lactic acid can also be added to the washing buffer to adjust the pH value of the washing buffer, and the concentration of lactic acid in the washing buffer is 0.01 to 1% (v/v), preferably 0.01 to 0.5% (v/v).

In one embodiment, the first washing buffer of the present case also uses guanidine thiocyanate (GuSCN), one of the most effective liquid separation agents, and the concentration of GuSCN is 0.5 to 3 M, preferably 1 to 2 M. The second washing buffer uses hexammine cobalt (III) chloride, and its concentration is 1 to 25 mM, preferably 1 to 5 mM. Since it is a tetravalent metal ion, it is a compaction agent that can selectively precipitate RNA and has a high RNA affinity, but can be easily stripped from RNA molecules during RNA purification without destroying RNA molecules.

The wash buffer uses 5 to 20% (v/v), preferably 5 to 10% (v/v) high molecular weight PEG8000 to effectively concentrate nucleic acids from the sample. The first wash buffer uses 0.5 to 2 M, preferably 0.5 to 1 M sodium chloride, and the second wash buffer uses 5 to 1000 mM, preferably 20 to 200 mM sodium chloride to help maintain the strength of the ionic medium and promote the release of cellular components into the solution. Ethylenediaminetetraacetic acid (EDTA) is a chelating agent used in the first wash buffer at concentrations of 5 to 25 mM that helps inactivate nucleases and helps prevent degradation of DNA and RNA.

As described above, the first washing buffer used in the magnetic bead-based nucleic acid extraction system of the present invention includes 0.5 to 3 M GuSCN, 5 to 20% (v/v) PEG8000, 0.5 to 2 M sodium chloride, 5 to 25 mM EDTA, and a non-ionic detergent selected from the group consisting of 0.25 to 4% (v/v) Ecosurf EH-9, 0.1 to 4% (v/v) Triton X-100, 0.25 to 4% (v/v) Tergitol 15-S-40 and a mixture thereof. The pH value of the first washing buffer is adjusted to 3 to 5 with 1 M HCl. Alternatively, 0.01 to 1% (v/v) lactic acid may be added to the first washing buffer to adjust the pH value. This case provides three sets of first cleaning buffer solutions A, B, and C, and their formulas are shown in Tables 4 to 6 below.

Table 4: First cleaning buffer A Element Concentration range Tris-HCl (pH 3 to 5) 10 to 100 mM PEG8000 5 to 20% (v/v) Sodium chloride 0.5 to 2 M EDTA 5 to 25 mM G JZ 0.5 to 3 M Lactic acid 0.01 to 1% (v/v) Ecosurf EH-9 0.25 to 4% (v/v)

Table 5: First cleaning buffer B Element Concentration range Tris-HCl (pH 3 to 5) 10 to 100 mM PEG8000 5 to 20% (v/v) Sodium chloride 0.5 to 2 M EDTA 5 to 25 mM G JZ 0.5 to 3 M Lactic acid 0.01 to 1% (v/v) Triton X-100 0.1 to 4% (v/v)

Table 6: First cleaning buffer C Element Concentration range Tris-HCl (pH 3 to 5) 10 to 100 mM PEG8000 5 to 20% (v/v) Sodium chloride 0.5 to 2 M EDTA 5 to 25 mM G JZ 0.5 to 3 M Lactic acid 0.01 to 1% (v/v) Tergitol 15-S-40 0.25 to 4% (v/v)

As described above, the second washing buffer used in the magnetic bead-based nucleic acid extraction system of the present invention includes 1 to 25 mM hexaaminecobalt (III) chloride, 5 to 20% (v/v) PEG8000, 5 to 1000 mM sodium chloride, and a non-ionic detergent selected from the group consisting of 0.25 to 2% (v/v) Ecosurf EH-9, 0.1 to 2% (v/v) Triton X-100, 0.25 to 2% (v/v) Tergitol 15-S-40 and a mixture thereof. The pH value of the second washing buffer is adjusted to 3 to 5 with 1 M HCl. Alternatively, 0.01 to 1% (v/v) lactic acid may be added to the second washing buffer to adjust the pH value. This case provides three sets of second cleaning buffer solutions A, B, and C, and their formulas are shown in Tables 7 to 9 below.

Table 7: Second cleaning buffer A Element Concentration range Tris-HCl (pH 3 to 5) 10 to 100 mM PEG8000 5 to 20% (v/v) Sodium chloride 5 to 1000 mM Hexaamminecobalt(III) chloride 1 to 25 mM Lactic acid 0.01 to 1% (v/v) Ecosurf EH-9 0.25 to 2% (v/v)

Table 8: Second cleaning buffer B Element Concentration range Tris-HCl (pH 3 to 5) 10 to 100 mM PEG8000 5 to 20% (v/v) Sodium chloride 5 to 1000 mM Hexaamminecobalt(III) chloride 1 to 25 mM Lactic acid 0.01 to 1% (v/v) Triton X-100 0.1 to 2% (v/v)

Table 9: Second cleaning buffer C Element Concentration range Tris-HCl (pH 3 to 5) 10 to 100 mM PEG8000 5 to 20% (v/v) Sodium chloride 5 to 1000 mM Hexaamminecobalt(III) chloride 1 to 25 mM Lactic acid 0.01 to 1% (v/v) Tergitol 15-S-40 0.25 to 2% (v/v)

The wash buffer releases the nucleic acids from the magnetic beads. When the salt concentration in the buffer is low, the nucleic acids can come into contact with water molecules, causing them to transform into a fully hydrated form during the wash phase.

The pH value of the washing buffer in this case is 8, which is provided by Tris-HCl. At this pH value, the binding between nucleic acid and magnetic beads is destroyed, causing the nucleic acid to be released from the magnetic beads.

In one embodiment, the optimal concentration of Tris-HCl (pH 8) is 1 to 100 mM, and the optimal concentration of EDTA is 1 to 25 mM. If necessary, the pH value can be adjusted to 8 with 50 mM HCl. The formula of the flushing buffer in this case is shown in Table 10 below.

Table 10: Flushing Buffer Element Concentration range Tris-HCl (pH 8) 1 to 100 mM EDTA 1 to 25 mM

The extraction protocol developed using the above buffer and magnetic beads is capable of extracting DNA and RNA separately or simultaneously from swab samples.

The system can be used with different types of magnetic beads with various coatings and particle sizes. The coating of the magnetic beads can be, but are not limited to, silica, polymer, carboxyl, or PEG, and the particle size of the magnetic beads ranges from 10 nm to 3 μm. The system can be used for manual purification or can be integrated into a device for automated preparation of nucleic acids from swab samples.

The present invention also provides a magnetic bead-based nucleic acid extraction method using the above buffer and magnetic beads. The following example illustrates the procedure of manually extracting nucleic acids in a 1.5 ml Eppendorf tube on a workbench using the lysis and binding buffer, the first washing buffer, the second washing buffer, and the washing buffer prepared according to the above embodiment.

1. Pipette 15 μl of magnetic beads into a 1.5 ml Eppendorf tube.

2. Add 600 μl of lysis buffer to the tube. Optionally, vortex or mix briefly with a pipette until a homogenous mixture is obtained.

3. Add 300 μl of sample to the lysis buffer containing magnetic beads and resuspend the entire mixture until a homogenous mixture is formed.

4. Allow the tube to stand at room temperature for 4 minutes. Continuous stirring/re-suspension is not necessary.

5. Place the tube on the magnetic stand for 30 seconds or until the supernatant turns clear, then carefully remove and discard the supernatant. (Note: The nucleic acid/magnetic bead complex will adhere to the side of the Eppendorf tube and have a visible dark gel-like appearance.)

6. Remove the tube from the magnet. Add 200 μl of the first wash buffer and resuspend the nucleic acid/magnetic bead complex until it is evenly dispersed.

7. Place the tube on the magnetic stand and incubate for 20 seconds or until the supernatant becomes clear. Carefully remove and discard the supernatant.

8. Remove the tube from the magnet. Add 200 μl of the second wash buffer and resuspend the nucleic acid/magnetic bead complex until it is evenly dispersed.

9. Place the tube on the magnetic stand and incubate for 20 seconds or until the supernatant becomes clear. Carefully remove and discard the supernatant.

10. Remove the tube from the magnetic stand. Resuspend the nucleic acid/magnetic bead complex in 80 μl of wash buffer until the suspension becomes homogeneous.

11. Incubate the mixture at 80°C for 2 minutes and then quickly centrifuge the tube to remove any liquid from the cap. Place the tube on a magnetic stand and incubate for 20 seconds or until the supernatant becomes clear.

12. Transfer the supernatant containing nucleic acids to a fresh tube for use in downstream reactions.

The following examples will illustrate the application of the magnetic bead-based nucleic acid extraction method of this case to extract nucleic acids from different microorganisms.

In the following examples, samples were prepared using viral transport medium (VTM) and swab simulation buffer. The formula of swab simulation buffer is shown in Table 11. The experimental sample buffer includes 250 μl VTM and 50 μl swab simulation buffer (hereinafter referred to as 300 μl experimental sample buffer).

Table 11: Swab Simulation Buffer Element Concentration Porcine mucin 2.5% (w/v) Human whole blood 1% (v/v) Sodium chloride 0.85% (w/v) Phosphate buffered saline (PBS) 1x glycerin 15% (v/v)

Example 1 illustrates the manual extraction of RNA from human coronavirus HCoV-OC43 using buffer set A, wherein buffer set A includes lysis and binding buffer A, first washing buffer A, second washing buffer A, and the present washing buffer. 1 pfu (plaque-forming unit, a unit of virus quantity) of HCoV-OC43 was added to 300 μl of experimental sample buffer, and then the above extraction procedure was performed using buffer set A. 10 μl of the obtained washing solution was used for qPCR reaction for target detection of HCoV-OC43. Figure 1 shows the amplification curve and Cq value of the qPCR analysis in Example 1. The results show that buffer set A can successfully extract HCoV-OC43 RNA without affecting the subsequent nucleic acid amplification and detection.

Example 2 illustrates the manual extraction of DNA from Staphylococcus aureus (SA) using buffer set A, wherein buffer set A includes lysis-binding buffer A, first wash buffer A, second wash buffer A, and the present wash buffer. 10^5 cfu (colony-forming unit, a unit of bacterial count) of Staphylococcus aureus was mixed into 300 μl of the experimental sample buffer, and then the above extraction procedure was performed using buffer set A. 10 μl of the obtained wash buffer was used in a qPCR reaction for target detection of Staphylococcus aureus. Figure 2 shows the amplification curve and Cq value of the qPCR analysis in Example 2. The results show that buffer set A can successfully extract the DNA of Staphylococcus aureus without affecting the subsequent nucleic acid amplification and detection.

Example 3 illustrates the manual extraction of DNA from human adenovirus type 3 (Ad3) using buffer set A, wherein buffer set A includes lysis and binding buffer A, first washing buffer A, second washing buffer A, and the present washing buffer. 2500 pfu of human adenovirus type 3 was added to 300 μl of experimental sample buffer, and then the above extraction procedure was performed using buffer set A. 10 μl of the obtained washing solution was used for qPCR reaction to perform target detection of human adenovirus type 3. FIG. 3 shows the amplification curve and Cq value of the qPCR analysis in Example 3. The results show that buffer set A can successfully extract human adenovirus type 3 DNA without affecting the subsequent nucleic acid amplification and detection.

Example 4 illustrates the manual extraction of RNA from human coronavirus HCoV-OC43 using buffer set B, wherein buffer set B includes lysis and binding buffer B, first washing buffer B, second washing buffer B, and the washing buffer of this case. 3 x 10^-4 pfu of HCoV-OC43 was added to 300 μl of experimental sample buffer, and then the above extraction procedure was performed using buffer set B. 10 μl of the obtained washing solution was used for qPCR reaction for target detection of HCoV-OC43. Figure 4 shows the amplification curve and Cq value of the qPCR analysis in Example 4. The results show that buffer group B can successfully extract HCoV-OC43 RNA without affecting the subsequent nucleic acid amplification and detection.

Example 5 illustrates the manual extraction of DNA from Staphylococcus aureus using buffer set B, wherein buffer set B includes lysis and binding buffer B, first washing buffer B, second washing buffer B, and the present washing buffer. 100 cfu of Staphylococcus aureus was mixed into 300 μl of experimental sample buffer, and then the above extraction procedure was performed using buffer set B. 10 μl of the obtained washing solution was used for qPCR reaction for target detection of Staphylococcus aureus. FIG. 5 shows the amplification curve and Cq value of qPCR analysis in Example 5. The results show that buffer set B can successfully extract DNA of Staphylococcus aureus without affecting the subsequent nucleic acid amplification and detection.

Example 6 illustrates the manual extraction of DNA from human adenovirus type 3 using buffer set B, wherein buffer set B includes lysis and binding buffer B, first washing buffer B, second washing buffer B, and the washing buffer of this case. 0.375 pfu of human adenovirus type 3 was added to 300 μl of experimental sample buffer, and then the above extraction procedure was performed using buffer set B. 10 μl of the obtained washing solution was used for qPCR reaction to perform target detection of human adenovirus type 3. FIG. 6 shows the amplification curve and Cq value of qPCR analysis in Example 6. The results show that buffer set B can successfully extract human adenovirus type 3 DNA without affecting subsequent nucleic acid amplification and detection.

Example 7 illustrates the manual extraction of RNA from human coronavirus HCoV-OC43 using buffer set C, wherein buffer set C includes lysis and binding buffer C, first washing buffer C, second washing buffer C, and the washing buffer of this case. 3 x 10^-4 pfu of HCoV-OC43 was added to 300 μl of experimental sample buffer, and then the above extraction procedure was performed using buffer set C. 10 μl of the obtained washing solution was used for qPCR reaction for target detection of HCoV-OC43. Figure 7 shows the amplification curve and Cq value of qPCR analysis in Example 7. The results show that buffer group C can successfully extract HCoV-OC43 RNA without affecting subsequent nucleic acid amplification and detection.

Example 8 illustrates the manual extraction of DNA from Staphylococcus aureus using buffer set C, wherein buffer set C includes lysis-binding buffer C, first washing buffer C, second washing buffer C, and the present washing buffer. 100 cfu of Staphylococcus aureus was mixed into 300 μl of experimental sample buffer, and then the above extraction procedure was performed using buffer set C. 10 μl of the obtained washing solution was used for qPCR reaction to perform target detection of Staphylococcus aureus. FIG. 8 shows the amplification curve and Cq value of qPCR analysis in Example 8. The results show that buffer set C can successfully extract DNA of Staphylococcus aureus without affecting subsequent nucleic acid amplification and detection.

Example 9 illustrates the manual extraction of DNA from human adenovirus type 3 using buffer set C, wherein buffer set C includes lysis and binding buffer C, first washing buffer C, second washing buffer C, and the washing buffer of this case. 0.375 pfu of human adenovirus type 3 was added to 300 μl of experimental sample buffer, and then the above extraction procedure was performed using buffer set C. 10 μl of the obtained washing solution was used for qPCR reaction to perform target detection of human adenovirus type 3. FIG. 9 shows the amplification curve and Cq value of the qPCR analysis in Example 9. The results show that buffer set C can successfully extract human adenovirus type 3 DNA without affecting the subsequent nucleic acid amplification and detection.

In addition to the PCR reaction, the compatibility of the manual extraction system of this case with the LAMP reaction was further tested. Example 10 illustrates the use of the buffer system of this case to manually extract RNA from the human coronavirus HCoV-OC43, wherein the extracted nucleic acid is detected using the LAMP reaction. 0.1 pfu of HCoV-OC43 was mixed into 300 μl of the experimental sample buffer, and then the above extraction procedure was performed. 10 μl of the obtained extract was used in the LAMP reaction for target detection of HCoV-OC43. Figure 10 shows the expansion curve and Cq value of the LAMP analysis in Example 10. The results show that the buffer system of this case is also compatible with the LAMP reaction.

Example 11 illustrates the manual extraction of both DNA and RNA from human coronavirus HCoV-OC43 and Staphylococcus aureus using the buffer system of this case. 0.1 pfu of HCoV-OC43 and 10^4 cfu of Staphylococcus aureus were co-mixed into 300 μl of experimental sample buffer, and then the above extraction procedure was performed. 10 μl of the obtained extract was used in qPCR reaction for target detection of HCoV-OC43 and Staphylococcus aureus respectively. Figures 11A and 11B show the expansion curve and Cq value of the qPCR analysis in Example 11. The results show that in addition to extracting DNA and RNA separately, the buffer system of this case can also be used for the simultaneous extraction of DNA and RNA.

Example 12 illustrates the manual extraction of RNA from different concentrations of human coronavirus HCoV-OC43 using the buffer system of this case. 1.5 x 10^-4 pfu, 10^-3 pfu, and 10^-2 pfu of HCoV-OC43 were respectively mixed into 300 μl of experimental sample buffer, and then the above extraction procedure was performed. 10 μl of the individual extracts were used in qPCR reactions for target detection of HCoV-OC43. Figure 12 shows the expansion curve and Cq value of the qPCR analysis in Example 12. The results show that the buffer system of this case can extract RNA from low concentrations of HCoV-OC43.

Example 13 illustrates the manual extraction of DNA from different concentrations of Staphylococcus aureus using the buffer system of this case. 15 cfu, 100 cfu, and 1000 cfu of Staphylococcus aureus were mixed into 300 μl of experimental sample buffer, and then the above extraction procedure was performed. 10 μl of the individual extracts were used in qPCR reactions for target detection of Staphylococcus aureus. Figure 13 shows the expansion curve and Cq value of the qPCR analysis in Example 13. The results show that the buffer system of this case can extract DNA from low concentrations of Staphylococcus aureus.

Example 14 illustrates the comparison of RNA extraction from 1.5 x 10^-4 pfu of human coronavirus HCoV-OC43 using the buffer system of this case and the industry-leading commercial kit. This case protocol was compared with the Katsura Viral DNA/RNA Respi Kit and the Macherey-Nagel (MN) NucleoMag Pathogen Kit. 10 μl of the individual extracts were used in qPCR reactions for target detection of HCoV-OC43. Figure 14 shows the expansion curves and Cq values of the qPCR analysis in Example 14, showing that this case protocol has the best performance, followed by the Katsura Viral DNA/RNA Respi Kit and the Macherey-Nagel (MN) NucleoMag Pathogen Kit.

Example 15 illustrates the comparison of DNA extraction from 15 cfu of Staphylococcus aureus using the buffer system of this case and the industry-leading commercial kit. This case protocol was compared with the Katsura Viral DNA/RNA Respi kit. 10 μl of the individual extracts were used in qPCR reactions for target detection of Staphylococcus aureus. Figure 15 shows the expansion curve and Cq value of the qPCR analysis in Example 15, and the results show that this case protocol has better performance than the Katsura Viral DNA/RNA Respi kit.

Figure 16 shows a comparison of the turnaround time of the nucleic acid extraction solution of the present solution and the industry-leading commercial kit. As shown in Figure 16, the turnaround time of the present solution is less than 10 minutes, which is the shortest compared to the industry-leading commercial kit.

Figure 17 shows a comparison of the features of the present invention with the industry-leading commercial kit. As shown in Figure 17, the present invention is alcohol-free, enzyme-free, and requires only room temperature storage. There are no pre-treatment or drying steps in the present invention, and no centrifugation is required. In addition, the present invention has the fewest total steps and the least total time of the compared kits.

On the other hand, the compatibility of the manual extraction system in this case with different types of magnetic beads was further tested. The different types of magnetic beads are shown in Table 12 below.

Table 12: Magnetic Bead Types Magnetic beads Coating Particle size A Carboxyl 10 nm B Carboxyl 20 nm C Carboxyl 200 nm D Silica, carboxyl modified 300-500 nm E Silicon dioxide 10 nm F Polyethylene glycol 1 (PEG1) 10 nm G Polymer 1-3 um H Carboxyl 0.8-1 μm I Silanol 0.2-2 μm J Silicon dioxide 2-10 μm

Figures 18A and 18B show the expansion curves and Cq values of qPCR analysis using different types of magnetic beads for nucleic acid extraction. The results show that the proposed protocol can be used for different types of magnetic beads with various coatings and particle sizes.

The present solution is also ideal for automated nucleic acid extraction. For example, the present solution can be applied to an automated nucleic acid extraction machine (M32 nucleic acid extraction machine, Katsura Biological Corp.). Figure 19 shows the yield standard curve of using Vircell RNA and the present solution on an automated nucleic acid extraction machine. The results show excellent linearity, which also proves that the present solution can be well integrated into an automated nucleic acid extraction device.

In addition, Figure 20 shows the yield comparison between the present solution and the industry-leading commercial kit Qiagen viral RNA kit using Vircell RNA on the automatic nucleic acid extraction machine M32. The results show that when using Vircell RNA for testing, the present solution has a good recovery rate compared to the industry-leading commercial kit.

Based on the above, this case demonstrates a novel nucleic acid extraction system with an originally developed buffer and related solutions to address the challenges encountered in previous technologies and meet the WHO requirements for POC testing, which are described by the acronym ASSURED standard, namely affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free, and deliverable to end-users.

Accordingly, the present invention provides a stable, user-friendly, environmentally friendly, effective and efficient nucleic acid extraction and purification system suitable for pathogens in swab samples. The system includes a buffer, magnetic beads and a scheme for extracting nucleic acids from swab samples. The buffer includes a lysis binding buffer, a washing buffer and an extraction buffer that are stable at room temperature. The buffer includes specially selected separation salts, detergents, precipitants, and other components required for ideal nucleic acid extraction. The system is capable of lysing the starting material, binding the nucleic acid to the magnetic beads, washing away inhibitors or debris, and extracting the nucleic acid from the magnetic beads. The protocol has been optimized to effectively extract DNA and RNA from viruses and bacteria in swab samples either individually or simultaneously.

Therefore, the magnetic bead-based nucleic acid extraction system of this case has the following advantages.

1. User-friendly and environmentally friendly

This case does not include any corrosive, carcinogenic, toxic, harmful or flammable ingredients that may have adverse effects on people who handle or use it and the environment in which it is stored and used. This case avoids the use of organic solvents commonly used in other DNA isolation procedures, including ethanol.

2. Cost-effective and easy to transport, store and use

The original buffers and beads used in this case are stable at room temperature. These buffers are specially formulated to not contain enzymes for cell lysis (such as proteinase K), especially some bacteria that require harsh lysis conditions and/or destroy nucleases released after cell lysis. Excluding temperature-sensitive components that usually need to be stored at -20°C, such as enzymes and carrier RNA/DNA, allows this system to remain stable during storage, transportation and use at room temperature.

3. Short turnaround time

In this case, magnetic beads are used as an extraction tool. Once nucleic acids are released from cell lysis, the magnetic beads specifically bind to the nucleic acids and hold them in the washing step to remove impurities. Under the magnetic force of the magnetic plate, the magnetic beads are separated from the solution very quickly, which can be completed within 30 seconds. Coupled with efficient cell lysis and washing, even manual extraction takes less than 10 minutes, while faster commercial kits take about half an hour.

4. General formula and workflow for extracting DNA and RNA from pathogens (including viruses and most bacteria) in swab samples

This kit is capable of extracting both DNA and RNA from separate samples or multiple pathogens in one sample. This is better than most commercial kits that are used to specifically extract RNA or DNA from a single type of pathogen (such as virus or bacteria).

5. Huge potential for automation and high throughput

Most commercially available nucleic acid extraction kits require the end user to provide a list of consumables and/or equipment, which can only be met by well-equipped laboratories and require an additional centrifugation step (such as column-based methods). Since magnetic bead separation technology is used in this case, the entire process of nucleic acid extraction can be completed in one container, such as a 1.5 mL Eppendorf tube used for manual extraction. With the help of a liquid handling system, fully automated and high-throughput sample processing can be achieved.

6. Performance is competitive with industry-leading commercial nucleic acid extraction kits

Comparison of the extraction efficiency of several commercial kits showed that this case demonstrated equivalent or better performance compared to the Katsura Viral DNA/RNA Respi Kit, Macherey-Nagel NucleoMag Pathogen Kit, and QIAamp Viral RNA Mini Kit.

Although the present invention has been described in detail by the above embodiments, it can be modified in various ways by those skilled in the art, but it does not deviate from the scope of protection of the attached patent application.

without

Figure 1 shows the expansion curve and Cq value of the qPCR analysis in Example 1. Figure 2 shows the expansion curve and Cq value of the qPCR analysis in Example 2. Figure 3 shows the expansion curve and Cq value of the qPCR analysis in Example 3. Figure 4 shows the expansion curve and Cq value of the qPCR analysis in Example 4. Figure 5 shows the expansion curve and Cq value of the qPCR analysis in Example 5. Figure 6 shows the expansion curve and Cq value of the qPCR analysis in Example 6. Figure 7 shows the expansion curve and Cq value of the qPCR analysis in Example 7. Figure 8 shows the expansion curve and Cq value of the qPCR analysis in Example 8. Figure 9 shows the expansion curve and Cq value of the qPCR analysis in Example 9. FIG. 10 shows the expansion curve and Cq value of LAMP analysis in Example 10. FIG. 11A and FIG. 11B show the expansion curve and Cq value of qPCR analysis in Example 11. FIG. 12 shows the expansion curve and Cq value of qPCR analysis in Example 12. FIG. 13 shows the expansion curve and Cq value of qPCR analysis in Example 13. FIG. 14 shows the expansion curve and Cq value of qPCR analysis in Example 14. FIG. 15 shows the expansion curve and Cq value of qPCR analysis in Example 15. FIG. 16 shows a comparison of the turnaround time of nucleic acid extraction solutions of the present solution and the industry-leading commercial kit. FIG. 17 shows a comparison of the features of the present solution and the industry-leading commercial kit. Figures 18A and 18B show the expansion curves and Cq values of qPCR analysis using different types of magnetic beads for nucleic acid extraction. Figure 19 shows the yield standard curve using Vircell RNA and our solution on an automated nucleic acid extraction machine. Figure 20 shows the yield comparison of Vircell RNA and our solution with the industry-leading commercial kit on an automated nucleic acid extraction machine.

Claims (8)

A magnetic bead-based nucleic acid extraction system, comprising: a plurality of magnetic beads; and an alcohol-free buffer system, comprising: a lysis-binding buffer, comprising 0.5 to 4 M guanidine thiocyanate, 5 to 20% (v/v) PEG8000, 10 to 1000 mM sodium citrate, and a non-ionic detergent selected from the group consisting of 0.5 to 4% (v/v) NP-40 substitute, 0.5 to 4% (v/v) Tergitol 15-S-40, 0.5 to 4% (v/v) Ecosurf EH-9 and a mixture thereof; a first washing buffer, comprising 0.5 to 3 M guanidine thiocyanate, 5 to 20% (v/v) PEG8000, 0.5 to 2 M sodium chloride, 5 to 25 mM EDTA, and a non-ionic cleaner selected from the group consisting of 0.25 to 4% (v/v) Ecosurf EH-9, 0.1 to 4% (v/v) Triton X-100, 0.25 to 4% (v/v) Tergitol 15-S-40 and mixtures thereof; a second washing buffer comprising 1 to 25 mM hexaamminecobalt(III) chloride, 5 to 20% (v/v) PEG8000, 5 to 1000 mM sodium chloride, and a non-ionic cleaner selected from the group consisting of 0.25 to 2% (v/v) Ecosurf EH-9, 0.1 to 2% (v/v) Triton X-100, 0.25 to 2% (v/v) Tergitol 15-S-40 and mixtures thereof; and a flushing buffer. A magnetic bead-based nucleic acid extraction system as described in claim 1, wherein the pH value of the lysis and binding buffer is 3 to 5. A magnetic bead-based nucleic acid extraction system as described in claim 1, wherein the pH value of the first washing buffer is 3 to 5. A magnetic bead-based nucleic acid extraction system as described in claim 1, wherein the first washing buffer further comprises 0.01 to 1% (v/v) lactic acid. A magnetic bead-based nucleic acid extraction system as described in claim 1, wherein the pH value of the second washing buffer is 3 to 5. A magnetic bead-based nucleic acid extraction system as described in claim 1, wherein the second washing buffer further comprises 0.01 to 1% (v/v) lactic acid. A magnetic bead-based nucleic acid extraction system as described in claim 1, wherein the pH value of the flushing buffer is 8. The magnetic bead-based nucleic acid extraction system as described in claim 1, wherein the washing buffer comprises 1 to 25 mM EDTA.

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